Antimicrobial Peptide Maculatin Research Articles

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.

Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis.

The Location of the Antimicrobial Peptide Maculatin 1.1 in Model Bacterial Membranes.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from the skin secretions of Australian tree frogs. In this work, the interaction of Mac1 with anionic phospholipid bilayers was investigated by NMR, circular dichroism (CD) spectroscopy, neutron reflectometry (NR) and molecular dynamics (MD). In buffer, the peptide is unstructured but in the presence of anionic (DPC/LMPG) micelles or (DMPC/DMPG/DHPC) bicelles adopts a helical structure. Addition of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC/LMPG micelle solution showed that the N-terminus is more exposed to the hydrophilic Gd-DTPA than the C-terminus in micelles. 2H and 31P solid-state NMR showed that Mac1 had a greater effect on the anionic lipid (DMPG). A deuterium labeled Mac1 used in NR experiments indicated that the AMP spanned across anionic (PC/PG) bilayers, which was compatible with MD simulations. Simulations also showed that Mac1 orientation remained transmembrane in bilayers and wrapped on the surface of the micelles regardless of the lipid or detergent charge. Thus, the peptide orientation appears to be more susceptible to curvature than charged surface. These results support the formation of transmembrane pores by Mac1 in model bacterial membranes.

The antimicrobial peptide maculatin self assembles in parallel to form a pore in phospholipid bilayers

Little is known experimentally about the detailed orientation of membrane-bound maculatin 1.1 (Mac1), an antimicrobial peptide from the skin secretions of Australian tree frogs. In this work multiple 15N-labelled or 2H-labelled Mac1 with dodecylphosphocholine (DPC) micelles and isotropic DMPC/DHPC (q = 0.5) bicelles were investigated by solution NMR, circular dichroism (CD) spectroscopy, neutron reflectometry and molecular dynamics (MD) simulations in explicit solvent. In buffer, the 15N-1H HSQC and CD spectra were indicative of the peptide being random coiled. In the presence of micelles or isotropic bicelles, a unique and helical peptide structure that was confirmed by CD was found. The titration of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC solution led to enhanced relaxation of all 15N labelled residues. The peptide N-terminus was more exposed to Gd-DTPA than the C-terminus in micelles, while only the Gly-4 and Ala-18 resonances were significantly reduced in the presence of isotropic bicelles. MD simulations of Mac1 fully inserted into a DPC micelle converged towards a solvent exposed orientation and a topology where Mac1 was wrapped around the DPC micelle with the more hydrophobic side facing inward. MD simulations of Mac1 fully inserted into a phosphatidylcholine (PC) bilayer converged towards a kinked transmembrane orientation with water molecules penetrating around Lys-8. A deuterium labelled Mac1 used in neutron reflectometry experiments suggested a preferred orientation in zwitterionic PC bilayers. These results give insight into the membrane disrupting activity of Mac1 against cell membranes.

Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

Antimicrobial peptides (AMPs) that target lipid membranes show promise as alternatives to conventional antibiotics. However, the molecular mechanisms of membrane perturbation, as most studies are performed in model systems and in-cell structural studies, have yet to be achieved. Solid-state NMR spectroscopy is a valuable technique to investigate peptide-membrane interactions and to determine the structure of peptides, but the short lifespan of bacteria, especially under magic angle spinning conditions, has not permitted in-cell structural studies. Here, we present the first dynamic nuclear polarization (DNP)-NMR in-cell studies of Escherichia coli bacteria incubated with the AMP maculatin 1.1 (Mac1) in combination with novel nitroxide spin-labeled peptides 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC)-[F3W]-Mac1 (MacW) and TOAC-TOAC-MacW. The in-cell 13C and 15N signal NMR enhancements, and 1H spin-lattice T1 relaxation times showed that TOAC-MacW and TOAC-TOAC-MacW performed better than the more hydrophilic biradical AMUPol used for DNP studies. Furthermore, the pores formed by the AMP increased the signal enhancements and decreased T1 values of specifically 13C- and 15N-labeled Mac1. This approach has a great potential for determining the first in situ structures of AMPs in bacteria.-Sani, M.-A., Zhu, S., Hofferek, V., Separovic, F. Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

Effect of phosphatidylcholine bilayer thickness and molecular order on the binding of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides (AMPs) interact directly with bacterial membrane lipids. Thus, changes in the lipid composition of bacterial membranes can have profound effects on the activity of AMPs. In order to understand the effect of bilayer thickness and molecular order on the activity of AMPs, the interaction of maculatin 1.1 (Mac1.1) with phosphatidylcholine (PC) model membranes composed of different monounsaturated acyl chain lengths between 14 and 22 carbons was characterised by dual polarisation interferometry (DPI) and 31P and 1H solid-state NMR techniques. The thickness and bilayer order of each PC bilayer showed a linear dependence on the acyl chain length. The binding of Mac1.1 exhibited a biphasic dependency between the amount of bound Mac1.1 and bilayer thickness, whereby the mass of bound peptide increased from C14 to C16 and then decreased from C16 to C22. Significant perturbation of 31P chemical shift anisotropy (CSA) values was only observed for DOPC (C18) and DEPC (C22), respectively. In the case of DEPC, the greater range in CSA indicated different headgroup conformations or environments in the presence of Mac1.1. Overall, the results indicated that there is a significant change in the bilayer order upon binding of Mac1.1 and this change occurred in a co-operative manner at higher concentrations of Mac1.1 with increasing bilayer thickness and order. Overall, an optimum bilayer thickness and lipid order may be required for effective membrane perturbation by Mac1.1 and increasing the bilayer thickness and order may counteract the activity of Mac1.1 and play a role in antimicrobial resistance to AMPs.

Spontaneous Formation of an Ensemble of Structurally Diverse Membrane Channel Architectures from a Single Antimicrobial Peptide Maculatin

Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitous component of the innate immune defence in all domains of life and play a key role in controlling the human microbiome. Many AMPs are known to selectively target and form pores in microbial membranes, killing a wide variety of pathogens at low micromolar concentrations. Fundamental questions remain, however, regarding the molecular mechanisms of membrane targeting, pore formation and function, and the extent to which poration contributes towards antimicrobial activity. Here we report an experimentally guided and validated unbiased long-timescale simulation methodology that yields the complete mechanism of spontaneous pore assembly in the membrane for the amphiphilic pore-forming AMP maculatin at atomic resolution. Rather than a single well defined pore, maculatin was found to form an ensemble of well defined temporarily functional channels that continuously form and dissociate in the membrane. All channels are formed from highly symmetric low-oligomeric assemblies of up to 8 membrane-spanning peptides that mimic integral membrane protein channels in structure. Each channel has a different architecture, functional lifetime, and ion conductivity and overall membrane permeabilization is dominated by higher order oligomers, which form ion channels that also conduct water at high rates. The highest order oligomers were also found to efficiently conduct small dyes. Channel architectures as well as their relative weighting in the ensemble are sensitive to minor mutations as well as variation of lipid tail length. All pores are formed spontaneously by the consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models postulated for AMPs. The diversity of channel architectures formed by a single sequence is remarkable and could explain why no sequence-function relationships in AMP sequences have been discovered to date. Structural ensembles formed by AMP may be the key to preventing bacterial resistance.

Localisation of the Antimicrobial Peptide Maculatin 1.1 in Lipid Bilayers using Solid-State NMR

Antimicrobial peptides that target membranes are an attractive alternative to classic antibiotics, since they do not require internalization nor target a specific stereo-structure, thus limiting development of bacterial resistance. Their mode of action involves the disruption of lipid membranes, leading to growth inhibition and ultimately death of the bacteria. However, the molecular details of the killing mechanism and, more particularly, the difference in potency observed between different bacterial strains, remain unclear. Structural information is crucial for defining the molecular mechanism by which these peptides recognize and interact with a particular lipid membrane. Solid-state NMR is a non-invasive tool that allows study of the structural details of lipid-peptide interactions. Explicitly, we have combined 2H, 31P, {19F}13C and {31P}13C REDOR solid-state NMR experiments to obtain the localisation of the antimicrobial peptide maculatin 1.1 (Mac1) in anionic lipid bilayers (PC/PG 3:1). Mac1 showed reduced dynamics of d54-DMPG acyl chain compared to d54-DMPC. 31P NMR experiments also showed more pronounced effect on DMPG than neutral DMPC headgroups. Penetration depth of the peptide was further probed by REDOR experiments where the distance between the 13C labelled peptide and the acyl chain mono-fluorinated DPPC lipid or the phosphorous headgroup was measured. Furthermore, solution NMR using isotropic bicelles with similar lipid composition showed a helical peptide structure for Mac1 and addition of paramagnetic agents confirmed a transmembrane insertion. Overall, Mac1 showed a greater affinity for the anionic DMPG, recruiting the lipid while spaning the bilayer in a transmembrane orientation.

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μM) than against E. coli (64 μM) and an intermediate cytotoxicity against hRBC (30 μM). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.

Proline Facilitates Membrane Insertion of the Antimicrobial Peptide Maculatin 1.1 via Surface Indentation and Subsequent Lipid Disordering

The role of proline in the disruption of membrane bilayer structure upon antimicrobial peptide (AMP) binding was studied. Specifically, 31P and 2H solid-state NMR and dual polarization interferometry (DPI) were used to analyze the membrane interactions of three AMPs: maculatin 1.1 and two analogs in which Pro-15 is replaced by Gly and Ala. For NMR, deuterated dimyristoylphosphatidylcholine (d54-DMPC) and d54-DMPC/dimyristoylphosphatidylglycerol (DMPG) were used to mimic eukaryotic and prokaryotic membranes, respectively. In fluid-phase DMPC bilayer systems, the peptides interacted primarily with the bilayer surface, with the native peptide having the strongest interaction. In the mixed DMPC/DMPG bilayers, maculatin 1.1 induced DMPG phase separation, whereas the analogs promoted the formation of isotropic and lipid-enriched phases with an enhanced effect relative to the neutral DMPC bilayers. In gel-phase DMPC vesicles, the native peptide disrupted the bilayer via a surface mechanism, and the effect of the analogs was similar to that observed in the fluid phase. Real-time changes in bilayer order were examined via DPI, with changes in bilayer birefringence analyzed as a function of the peptide mass bound to the bilayer. Although all three peptides decreased the bilayer order as a function of bound concentration, maculatin 1.1 caused the largest change in bilayer structure. The NMR data indicate that maculatin 1.1 binds predominantly at the surface regions of the bilayer, and both NMR and DPI results indicate that this binding leads to a drop in bilayer order. Overall, the results demonstrate that the proline at residue 15 plays a central role in the membrane interaction of maculatin 1.1 by inducing a significant change in membrane order and affecting the ability of the bilayer to recover from structural changes induced by the binding and insertion of the peptide.

Immune evasion or avoidance: Fungal skin infection linked to reduced defence peptides in Australian green-eyed treefrogs, Litoria serrata

Many parasites and pathogens suppress host immunity to maintain infection or initiate disease. On the skin of many amphibians, defensive peptides are active against the fungus Batrachochytrium dendrobatidis (Bd), the causative agent of the emerging infectious disease chytridiomycosis. We tested the hypothesis that infection with the fungus may be linked to lower levels of defensive peptides. We sampled both ambient (or constitutive) skin peptides on the ventral surface immediately upon capture, and stored skin peptides induced from granular glands by norepinephrine administration of Australian green-eyed treefrogs, Litoria serrata. Upon capture, uninfected frogs expressed an array of antimicrobial peptides on their ventral surface, whereas infected frogs had reduced skin peptide expression. Expression of ambient skin peptides differed with infection status, and antimicrobial peptides maculatin 1.1 and 2.1 were on average three times lower on infected frogs. However, the repertoire of skin peptides stored in granular glands did not differ with infection status; on average equal quantities were recovered from infected and from uninfected frogs. Our results could have at least two causes: (1) frogs with reduced peptide expression are more likely to become infected; (2) Bd infection interferes with defence peptides by inhibiting release or causing selective degradation of peptides on the skin surface. Immune evasion therefore may contribute to the pathogenesis of chytridiomycosis and a mechanistic understanding of this fungal strategy may lead to improved methods of disease control.

Multiscale Simulations of the Antimicrobial Peptide Maculatin 1.1: Water Permeation through Disordered Aggregates

The antimicrobial peptide maculatin 1.1 (M1.1) is an amphipathic α-helix that permeabilizes lipid bilayers. In coarse-grained molecular dynamics (CG MD) simulations, M1.1 has previously been shown to form membrane-spanning aggregates in DPPC bilayers. In this study, a simple multiscale methodology has been applied to allow sampling of important regions of the free energy surface at higher resolution. Thus, by back-converting the CG configurations to atomistic representations, it is shown that water is able to permeate through the M1.1 aggregates. Investigation of aggregate stoichiometry shows that at least six peptides are required for water permeation. The aggregates are dynamically disordered structures, and water flux occurs through irregular, fluctuating channels. The results are discussed in relation to experimental data and other simulations of antimicrobial peptides.

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH(2)) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC-dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC-DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.

Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide–lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.

Disentanglement of Heterogeneous Dynamics in Mixed Lipid Systems

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of 31P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.

Neutron Reflectivity Studies of the Antimicrobial Peptide Maculatin 1.1 in Supported Lipid Bilayers

Maculatin 1.1 is a member of a small group of α-helical antimicrobial peptides isolated from the skin secretions of Australian amphibians. Previous studies using solid-state NMR, surface plasmon resonance, quartz crystal microbalance, calorimetry and fluorescence measurements have revealed that this peptide acts via different mechanisms dependent on concentration and membrane charge, which lead to the disruption of bacterial membranes. Neutron reflection is a method that provides information about the distribution of materials on the axis perpendicular to a membrane surface (the z-axis). Neutron scattering can discriminate between hydrogen and its isotope deuterium, making neutron reflection a powerful tool for dissecting how lipid, protein and solvent relate to one another along the z-axis and provides a method in which certain components can be highlighted or made invisible by choosing the correct isotopic contrast. In this case deuterated lipid chains and a hydrogenated peptide were used. The interaction of maculatin 1.1 in solid-supported lipid bilayers that mimic the essential charge characteristics of eukaryotic (neutral) and prokaryotic (anionic) membranes were studied using neutron reflection. The peptide was found to bind to both bilayers but the action of the peptide was different between each bilayer. When maculatin 1.1 bound to neutral bilayers the bilayer structure was lost and lipid order diminished whereas when maculatin 1.1 bound to negatively charged bilayers lipid order increased and the bilayer structure was retained suggesting that maculatin 1.1 formed pores when bound to negatively charged bilayers.

Interactions of the Antimicrobial Peptide Maculatin 1.1 and Analogues with Phospholipid Bilayers

The interactions of the antimicrobial peptide, maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH2) and two analogues, with model phospholipid membranes have been studied using solid-state NMR and circular dichroism spectroscopy. Maculatin 1.1 and the P15G and P15A analogues displayed minimal secondary structure in water, but with zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles displayed a significant increase in α-helical content. In mixed phospholipid vesicles of DMPC and anionic dimyristoylphosphatidylglycerol (DMPG), each peptide was highly structured with ~80% α-helical content. In DMPC vesicles, the native peptide displayed moderate head group interaction and significant perturbation of the lipid acyl chains. In DMPC/DMPG vesicles, maculatin 1.1 promoted formation of a DMPG-enriched phase and moderately increased disorder towards acyl chain ends of DMPC in the mixed bilayer. Both analogues showed reduced phospholipid head group interactions with DMPC but displayed significant interactions with the mixed lipid system. These effects support the preferential activity of these antimicrobial peptides for bacterial membranes.